Norbelladine biosynthesis

Oxidative phenolic coupling. Biosynthesis of the alkaloid narwedine (3) is known to involve oxidative phenolic coupling of norbelladine derivatives (1), but the usual oxidants for such coupling in vitro convert 1(R = H) into the oxomaritidine skeleton (4) rather than 3. A new biomimetic synthesis of 3 involves the palladacycle 2, formed by reaction of 1(R = CH3) with Li2PdCl4, which is known to form complexes with allylic amines or sulfides (8,176-177). Oxidation of 2 with thallium(III) trifluoroacetate effects the desired coupling to give 3. 

From L-tyrosine, or alternatively from L-phenylalanine, there is one further alkaloid biosynthesis pathway. This is the galanthamine pathway (Figure 38). Galanthamine synthesizes with tyramine, norbelladine, lycorine, crinine, N-demethylnarwedine and Al-demethylgalanthamine. Schiff base and reduction reaction, oxidative coupling and enzyme NADPH and SAM activity occur in this pathway. Schiff base is a reaction for the ehmination of water in formation with the C—N bonds process. 

In daffodil plants, galanthamine (147) is biosynthesized from the aminophenols 397, 398, and 399 but not from 343 which is, however, in the same plants a good precursor of haemanthamine (318) and lycorine (1) (Vol. XI, p. 397). These experimental results were interpreted as proving the existence of a definite order of methylation of norbelladine (399) during the biosynthesis of 147. Thus, methylation takes place in the first instance at nitrogen to give 398, later converted into 397. 

Although norbelladine was shown not to be a precursor of galanthamine (101) in King Alfred daffodils, an incorporation of this compound with labels as shown (103), comparable to that for lycorine (104), has been obtained for galanthamine in Leucojum aestivum As expected, the lycorine showed loss of half its tritium. On the other hand, no loss of tritium was apparent in the galanthamine. The latter result suggested that in the biosynthesis of galanthamine conversion of (105) to narwedine (100) was either not reversible or, if so, enzy-mically controlled. 

Thus, tyramine and protocatechuic aldehyde -or derivatives of the latter- are logical components for the biosynthesis of the precursor norbelladine (85). This reaction occupies a pivotal position since it represents the entry of primary metabolites into a secondary metabolic pathway. The junction of the amine and the aldehyde results in a Schiff s base, two of which have been isolated up to now craugsodine (114) and isocraugsodine (115). The existence of Schiff s bases in nature as well as their easy conversion into the different ring-systems of the Amaryllidaceae alkaloids allow the presumption that the initial postulate about this biosynthetic pathway was correct. 

Key intermediates in the biosynthesis of the crinine alkaloids are norbelladiene and 4 -0-methylnorbelladine (90). It became clear, however, as the result of experiments with doubly labelled precursors that neither these compounds nor 3 -0-methyl-, iV-methyl-, or 3 -OiV-dimethyl-norbelladine were directly involved in mesembrine biosynthesis the moderate levels of incorporation observed were found to be the result of prior fragmentation of the precursors. In an ultimate test... 

Tyramine and protocatechuic aldehyde or its derivatives are logical components for the biosynthesis of the precursor norbelladine (93). This pivotal reaction... 

Plant 0-methylation reactions are common transformation in the biosynthesis of alkaloids and are most often catalyzed by 5 -adenosyl-L-methionine (SAM)-dependent methyltransferases (MTs) [62-72], Thus, norbelladine must be 4 -0-methylated to form 4 -0-methylbelladine, a central intermediate from which multiple biosynthetic pathways lead to various structural types of AAs (Figures 1-2). 

In daffodil plants, galanthamine is biosynthesized from compounds 19-21, but not from norbelladine (5) [which is a good precursor of haemanthamine (14) and lycorine (8) in the same plants] (Fig. 33.6). These experiments suggest a definite order of methylation of norbelladine (5) during the biosynthesis of galathamine. In feeding experiments with the plant Leucojum aestivum, [2,4- H2 C-0-methyl]0-methylnorbelladine (13) is a precursor of galanthamine (18) that retains the same ratio as in the precursor. There... 

With the failure to demonstrate that norbelladine or its relatives plays a role in the biosynthesis of the mesembrine alkaloids, a reevaluation led to a modified approach in which attempts to identify the sequence of occurrence of the post-tyrosine and post-phenylalanine intermediates were made. There is now a substantial body of information available to suggest that phenylalanine and tyrosine have separate metabolic roles in plants belonging to the order Dictolyoden. Not only do they lack the enzyme phenylalanine hydroxylase (phenylalanine 4-monooxygenase) which is necessary for the conversion of phenylalanine to tyrosine, but the metabolic pathways of these two amino acids are generally quite different in secondary metabolism (70). Phenylalanine is involved in initial conversion to cinnamic acid and subsequent transformation to structural units of the so-called phenyl-propanoid pathway, which include Ar—C3, Ar—C2, and Ar—Cj structural entities. On the other hand, the role of tyrosine in the biosynthesis of secondary metabolities is most frequently seen as the precursor of Ar—Cj—N and Cg—C2—N units, and somewhat less frequently, as Ar—C2 and Q—C2 units. 

Studies on the biosynthesis of Amaryllidaceae alkaloids initiated as early as the 1960s. In 1957, Barton and Cohen proposed for the first time that norbelladine 2 or its congener alkaloid was probably the original precursor of other structurally diverse Amaryllidaceae alkaloids [103]. The primary metabolic processes leading to the Amaryllidaceae alkaloids include mainly (a) intramolecular phenol... 

Further experimental results established norbelladine 6.180) and some of its methylated derivatives (clearly not others) as key biosynthetic intermediates in the biosynthesis of, e.g., lycorine 6.185), haemanthamine 6.187) and galanthamine 6.190) [125-128, 132, 133]. As elsewhere (see Section 6.3) hydroxy-groups ortho and/or para to sites of new bond formation between aromatic rings are essential for biosynthesis to proceed, a telling set of examples in support of the phenol-oxidative coupling hypothesis. Of further interest is the reported isolation of an enzyme, from a plant of the Amaryl-lidaceae, which, when incubated with norbelladine and 5-adenosylmethionine (source of methyl groups), yielded almost entirely the 0-methylnorbelladine, 6.181), that is involved in alkaloid biosynthesis [134]. 

The third group of alkaloids which arise from norbelladine 6.180) this time by para-para phenol oxidative coupling, is exemplified by haemanthamine 6.187)., and biosynthesis is proved to be by way of compounds of type 6.186). Haemanthamine 6.187) shows an extra hydroxy-group at C-11, which has been shown to arise by hydroxylation with normal retention of configuration [139, 140]. 

The group of alkaloids exemplified by mesembrine (6.202), shows a structural kinship with Amaryllidaceae alkaloids of the haemanthamine (6.187) type. However, the only aspect of biosynthesis common to these two groups of alkaloids is their origin in phenylalanine and tyrosine results, crucially with doubly labelled precursors, showed that various norbelladine (6.180) derivatives were only incorporated after fragmentation [148]. 

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